HPLC is widely used to separate analytes in liquid samples. The sample is injected in an eluent into a chromatography column. The sample components are separated by one or more mechanisms including sorption, size exclusion, ion exchange or other interactions with the chromatography packing. The sample components are then detected by any conventional detector, e.g., UV, fluorescence or conductivity.
Silica-based RP (reverse phase) packing materials are commonly used in chromatography columns. They contain unreacted silanol groups on the silica surfaces. Residual silanol sites can interact with polar analytes, especially with basic compounds via ion exchange, hydrogen bonding and dipole-dipole mechanisms. These interactions can create problems ranging from increased retention to excessive tailing and irreversible adsorption of the sample. These undesirable effects can be minimized by using ultra pure silica and maximizing the surface coverage with hydrophobic ligands and endcapping reagents. However, highly aqueous mobile phases are often required to conduct separation of hydrophilic analytes by RP HPLC. Conventional RP C18 columns show a reversible loss of retention when operated under these conditions. The rate and degree of retention loss can vary greatly among different columns. A common explanation for this effect is that the hydrophobic alkyl chains are not wettable and appear to fold down on the silica surface to avoid the exposure to hydrophilic media. In this folded or matted-down state, the alkyl chains have reduced surface area for hydrophobic interaction with the solutes resulting in loss of retention. Reid, T. S.; Henry, R. A., American Laboratory 24-28 (July 1999).
In order to achieve the compatibility of RP C18 HPLC columns with highly aqueous mobile phases and to improve the peak shape of basic analytes, polar-embedded phases have been suggested. Majors, R. E., LC-GC 19:272-288 (2001). These phases are primarily hydrophobic but have hydrophilic groups incorporated near the silica surface. A benefit of this modification is that the polar-embedded phases exhibit typical reversed-phase behavior with water-organic solvent mixtures and can function well in highly aqueous environment with little or even no organic modifier present. The hydrophilic moieties also can impart different selectivity and retention characteristics compared to conventional reversed-phase materials. Polar-embedded phases have improved tailing characteristics for basic analytes due to shielding effect of the incorporated polar groups on residual silanols. Polar groups mask undesirable silanol activity and prevent hydrophilic analytes from being retained via interaction with underivatized silica surface.
In the early 1990's, conventional C18 and C8 silica stationary phases continued yielding large tailing factors for basic analytes. At that time, several research groups reported a novel type of bonded phases in which a polar functional group was embedded into the alkyl chains. These materials were found to reduce silanol interactions with basic analytes. The most commonly used polar groups were amide, urea, ether and carbamate functionalities.
Early reported materials were prepared by a two-step surface modification. Feibush, B. EP-A-0386926 (1990). In step one, the silica support was bonded with an aminopropyl silane. In step two, the amino groups reacted with an acid chloride to form amide linkages. This first generation methodology suffers from poor surface reproducibly over two successive reactions. Functionalized surface has a mixture of derivatized and underivatized amino groups. Therefore, the resulting phases exhibited undesirable ion exchange interactions due to the presence of unreacted basic sites.
Later, materials were prepared by one-step surface modification, where polar functional groups were built into a silane molecule. Neue, U. D.; Niederländer, C. L.; Perterson, J. S., EP-B-0579102 (1993). A single surface reaction with silane yields only one ligand structure with no anion exchange functionality being present.